1. Field of the Invention
This invention relates to improvements in methods and apparatus for differentiating and enumerating the various constituent subpopulations or types of cells in a whole blood sample.
2. Discussion of the Prior Art
In diagnosing different illnesses and disease states, it is common to analyze a patient's peripheral blood to differentiate and enumerate the various types of constituent blood cells and platelets, as well as to determine certain parameters or characteristics thereof. The respective concentrations and relative percentages of the different types of blood cells are highly indicative of certain diseases and the extent to which such diseases have spread. In general, a sample of whole blood comprises various types of cells, both blood cells and non-blood cells (e.g., tumor cells, bacteria, etc.) suspended in a liquid medium or plasma. The blood cells are three basic types, namely, red cells (erythrocytes), white cells (leukocytes), and platelets. Depending on the level of maturity, red cells are often further classified into three subsets, namely, nucleated red blood cells (NRBC's), reticulated red cells ("retics"), and mature red blood cells (RBC's). Mature white cells, on the other hand, fall into one of five different subsets, namely, monocytes, lymphocytes, eosinophils, neutrophils and basophils. Each of the white cell subsets can be further classified into subclasses based on their respective level of maturity, activation or abnormality. Typically, the number of red cells outnumber the total number of white cells by about 1000:1. Platelets, which are of interest at least from the standpoint that they control the ability of the blood to clot, are of three general types, mature platelets, reticulated platelets and large platelets.
In addition to determining the respective concentrations and relative percents of each of the above cell types and subsets, a thorough analysis of a blood sample will also provide information regarding various blood parameters and cell characteristics including, for example, the hemoglobin (Hgb) concentration, hematocrit value (Hct), mean cell volume (MCV), the total number of red and white cells and platelets per unit volume, the distribution width of red cells (RDW) and platelets (PDW), etc.
The detection and enumeration of most of the above cell types, as well as a determination of the above cell parameters, can be accomplished by using any one of several commercially available hematology instruments. Such instruments include Beckman Coulter's GEN.circle-solid.S.TM., STKS.TM., and MAXM.TM. Hematology Instruments; Abbott Laboratories' Cell Dyne 3000/4000 Hematology Instruments; and Toa's Sysmex Series of Hematology Instruments. In automatically acquiring data on each cell type, all of the above-mentioned hematology instruments use at least two discrete cell-analyzing transducers. One (or more) of these transducers operates to acquires data useful in differentiating and enumerating the five different types of white cells, and another transducer is dedicated to counting and sizing of red cells, white cells and platelets in a precise volume of sample. The respective outputs of the multiple transducers are processed by a central processing unit to provide an integrated cell analysis report. In the Beckman Coulter instruments, an electro-optical flow cell (transducer) produces signals indicative of the respective volume (V), electrical conductivity (C) and light scattering (S) properties of each white cell passing therethrough to provide a "five-part differential" of the five white cell types. Additional transducers operate on the well known Coulter Principle, one serving to count red cells and platelets in a highly diluted sample, and others serve to count white cells in a lysed sample. Information from the three transducers is processed and, in some cases, correlated (e.g., by multiplying the relative percentage of each white cell subset, as obtained from the electro-optical flow cell, by the absolute number of white cells counted by the Coulter transducer) to provide information about each cell type or subset, e.g., the concentration (number per unit volume) of each white cell subset in the whole blood sample being analyzed. In the Abbott instruments, the five-part diff information is provided by an optical flow cell that detects only light scatter and light polarization information. Here, again, a pair of additional transducers operating on the Coulter Principle serves to size and count white cells, red cells and platelets. The respective outputs of the two transducers are correlated with each other to report information on different cell types and subsets. In the Toa instruments, the five-part differential information is provided by a pair of electrical flow cells (Coulter transducers) that measure only the cell's DC volume and RF conductivity. Different lysing reagents are used to differentially process two or more aliquots of the blood sample, prior to passage through the two transducers. A third Coulter transducer operates to detect and count red cells and platelets. As in the Beckman Coulter and Abbott instruments, the respective outputs of the several transducers are correlated to provide the five-part differential information.
As indicated above, conventional hematology instruments, while being capable of differentiating and enumerating the vast majority of cell types and subsets in a peripheral blood sample, cannot readily differentiate all subsets of cells, particularly those that are abnormal or immature. An "extended differential" measurement by which these abnormal and immature cells may be detected and counted can be made manually by first producing a blood-smear of a sample of interest on a glass microscope slide, staining the smear with a dye to enable the cells to be visualized, whereby abnormal or immature cells of interest can be visually differentiated from other cells, and then examining the resulting stained blood-smear under a microscope. Alternatively, some blood types of an extended differential measurement can be detected using a conventional flow cytometer. In such an instrument, a blood sample that has been previously prepared, e.g., by either (1) mixing the sample with fluorochrome-labeled monoclonal antibodies or the like which serve to selectively "tag" certain cells of interest, or (2) mixing the sample with a fluorescent stain adapted to selectively mark cells of interest, is passed through an optical flow cell. As each cell in the sample passes through the flow cell, it is irradiated with a beam of photons adapted to excite the fluorescent material associated with the cells of interest. Fluorescent radiation emitted by each of the labeled cells, together with radiation scattered by each cell is detected and used to differentiated the cells of interest from other cells in the sample. Commercial, stand-alone, flow cytometers are made by Beckman Coulter, Toa Medical Electronics, Cytomation, Bio-Rad, and Becton Dickinson. It is known in the prior art to integrate flow cytometers and hematology instruments into a single automated laboratory system in which blood samples are automatically advanced along a track past these different instruments. As sample-containing vials pass each instrument, a blood sample is aspirated from each vial and analyzed by the instrument. Instrument systems combining discrete hematology and flow cytometry instruments are commercially available from Beckman Coulter and Toa Medical Electronics, reference being made to Toa's HST Series.
In U.S. Pat. Nos. 5,631,165 and 5,565,499, an attempt is made to fully integrate the respective functions of hematology and flow cytometry instruments into a single instrument. Such an instrument comprises a plurality of transducers, including an optical flow cell adapted to make fluorescence and multiangle light scatter measurements, an electrical impedance-measuring transducer (a Coulter transducer), and a colorimeter for measuring the overall hemoglobin content of a blood sample. The respective outputs from these transducers are processed and correlated to produce a report on red, white and fluorescent cells.
As suggested above, the requirement to correlate the respective outputs of multiple transducers in order to report certain characteristics of a cell type or subset can, under certain circumstances, be problematic in that it introduces an uncertainty in the analytical results. The validity of the requisite correlation step presupposes that the sample processed by one transducer is identical in content to that processed by the other transducer(s). This may not always be the case. Ideally, all of the measurements made on a cell should be made simultaneously by the same transducer. In such a case, there would be no need to correlate data from independent or separate transducers. Further, the simultaneous measurement of multiple parameters on a single cell using a single transducer enables a multidimensional cell analysis that would not be possible using separate transducers, or even using a single transducer when the parameter measurements are spatially separated in time.
The desirability of using a single electro-optical transducer to simultaneously measure the volume (V), conductivity (C), light scatter (S) and fluorescence (F) of a single cell has been suggested in the prior art. As noted above, such a transducer offers the advantage of making all measurements simultaneously on the same cell, rather than making some measurements on one cell with one transducer, making other measurements on another cell of the same type using another transducer, and then attempting to correlate the results from the two transducers to draw certain conclusions about the cell sample. In a disclosure by Thomas et al., Journal of Histochemistry and Cytochemistry, Vol. 25, No. 7, pp. 827-835 (1977), an automated multiparameter analyzer for cells (AMAC) is proposed. Such an analyzer comprises a single transducer adapted to simultaneously measure four different cell characteristic, namely, the above-noted V,C,S and F characteristics. In the Thomas et al. article, two different electro-optical flow cells are disclosed, one flow cell (the AMAC III) having a flow passageway of square transverse cross-section, measuring 100 microns on each side, and the other flow cell (AMAC IV) having a circular transverse cross-section. While the AMAC IV flow cell affords certain advantages in terms of manufacturability, the AMAC III flow cell was preferred due to its flat face geometry and its inherent ability to reduce optical aberrations, thereby better enabling the beam of excitation radiation to be focused on the cell path, and better enabling cell fluorescence to be coupled to a photo-detector. Though proposed for making simultaneous measurements of V, C, S and F, there is no evidence that either the AMAC III or IV flow cells was ever used for making such measurements. In the Thomas et al. article, for example, it is noted that an RF measurement (which is used in determining the "opacity" parameter) conducted on beads passing through the AMAC III flow cell gave rise to imprecise results. These results were attributable to a signal-to-noise problem associated with the relatively large cross sectional area (100.times.100 microns) of the flow cell. Further, it is apparent that light scattering measurements were never conducted using the AMAC flow cells. Thus, though others have proposed a single VCSF transducer for making simultaneous measurements on each cell, no one has either reduced such a concept to practice or has provided a technically enabling disclosure for doing so.